Biomedical subjects

Eric D Green

Publications and source records attributed to Eric D Green.

At least 37 records · Page 2Linked to original sources

PubMed2005

An initial strategy for the systematic identification of functional elements in the human genome by low-redundancy comparative sequencing.

With the recent completion of a high-quality sequence of the human genome, the challenge is now to understand the functional elements that it encodes. Comparative genomic analysis offers a powerful approach for finding such elements by identifying sequences that have been highly conserved during evolution. Here, we propose an initial strategy for detecting such regions by generating low-redundancy sequence from a collection of 16 eutherian mammals, beyond the 7 for which genome sequence data are already available. We show that such sequence can be accurately aligned to the human genome and used to identify most of the highly conserved regions. Although not a long-term substitute for generating high-quality genomic sequences from many mammalian species, this strategy represents a practical initial approach for rapidly annotating the most evolutionarily conserved sequences in the human genome, providing a key resource for the systematic study of human genome function.

Animals↗

PubMed2005

Novel human alpha1a-adrenoceptor single nucleotide polymorphisms alter receptor pharmacology and biological function.

We identified nine naturally-occurring human single nucleotide polymorphisms (SNPs) in the alpha(1a)-adrenoceptor (alpha(1a)AR) coding region, seven of which result in amino acid change. Utilizing rat-1 fibroblasts stably expressing wild type alpha(1a)AR or each SNP at both high and low levels, we investigated the effect of these SNPs on receptor function. Compared with wild type, two SNPs (R166K, V311I) cause a decrease in binding affinity for agonists norepinephrine, epinephrine, and phenylephrine, and also shift the dose-response curve for norepinephrine stimulation of inositol phosphate (IP) production to the right (reduced potency) without altering maximal IP activity. In addition, SNP V311I and I200S display altered antagonist binding. Interestingly, a receptor with SNP G247R (located in the third intracellular loop) displays increased maximal receptor IP activity and stimulates cell growth. The increased receptor signaling for alpha(1a)AR G247R is not mediated by altered ligand binding or a deficiency in agonist-mediated desensitization, but appears to be related to enhanced receptor-G protein coupling. In conclusion, four naturally-occurring human alpha(1a)AR SNPs induce altered receptor pharmacology and/or biological activity. This finding has potentially important implications in many areas of medicine and can be used to guide alpha(1a)AR SNP choice for future clinical studies.

Adrenergic alpha-Agonists↗

PubMed2005

Comparative sequencing provides insights about the structure and conservation of marsupial and monotreme genomes.

Sequencing and comparative analyses of genomes from multiple vertebrates are providing insights about the genetic basis for biological diversity. To date, these efforts largely have focused on eutherian mammals, chicken, and fish. In this article, we describe the generation and study of genomic sequences from noneutherian mammals, a group of species occupying unusual phylogenetic positions. A large sequence data set (totaling >5 Mb) was generated for the same orthologous region in three marsupial (North American opossum, South American opossum, and Australian tammar wallaby) and one monotreme (platypus) genomes. These ancient mammalian genomes are characterized by unusual architectural features with respect to G + C and repeat content, as well as compression relative to human. Approximately 14% and 34% of the human sequence forms alignments with the orthologous sequence from platypus and the marsupials, respectively; these numbers are distinctly lower than that observed with nonprimate eutherian mammals (45-70%). The alignable sequences between human and each marsupial species are not completely overlapping (only 80% common to all three species) nor are the platypus-alignable sequences completely contained within the marsupial-alignable sequences. Phylogenetic analysis of synonymous coding positions reveals that platypus has a notably long branch length, with the human-platypus substitution rate being on average 55% greater than that seen with human-marsupial pairs. Finally, analyses of the major mammalian lineages reveal distinct patterns with respect to the common presence of evolutionarily conserved vertebrate sequences. Our results confirm that genomic sequence from noneutherian mammals can contribute uniquely to unraveling the functional and evolutionary histories of the mammalian genome.

Animals↗

PubMed2005

Isolation and analysis of candidate myeloid tumor suppressor genes from a commonly deleted segment of 7q22.

Monosomy 7 and deletions of 7q are recurring leukemia-associated cytogenetic abnormalities that correlate with adverse outcomes in children and adults. We describe a 2.52-Mb genomic DNA contig that spans a commonly deleted segment of chromosome band 7q22 identified in myeloid malignancies. This interval currently includes 14 genes, 19 predicted genes, and 5 predicted pseudogenes. We have extensively characterized the FBXL13, NAPE-PLD, and SVH genes as candidate myeloid tumor suppressors. FBXL13 encodes a novel F-box protein, SVHis a member of a gene family that contains Armadillo-like repeats, and NAPE-PLD encodes a phospholipase D-type phosphodiesterase. Analysis of a panel of leukemia specimens with monosomy 7 did not reveal mutations in these or in the candidate genes LRRC17, PRO1598, and SRPK2. This fully sequenced and annotated contig provides a resource for candidate myeloid tumor suppressor gene discovery.

Base Sequence↗

PubMed2005

Detection of potential GDF6 regulatory elements by multispecies sequence comparisons and identification of a skeletal joint enhancer.

The identification of noncoding functional elements within vertebrate genomes, such as those that regulate gene expression, is a major challenge. Comparisons of orthologous sequences from multiple species are effective at detecting highly conserved regions and can reveal potential regulatory sequences. The GDF6 gene controls developmental patterning of skeletal joints and is associated with numerous, distant cis-acting regulatory elements. Using sequence data from 14 vertebrate species, we performed novel multispecies comparative analyses to detect highly conserved sequences flanking GDF6. The complementary tools WebMCS and ExactPlus identified a series of multispecies conserved sequences (MCSs). Of particular interest are MCSs within noncoding regions previously shown to contain GDF6 regulatory elements. A previously reported conserved sequence at -64 kb was also detected by both WebMCS and ExactPlus. Analysis of LacZ-reporter transgenic mice revealed that a 440-bp segment from this region contains an enhancer for Gdf6 expression in developing proximal limb joints. Several other MCSs represent candidate GDF6 regulatory elements; many of these are not conserved in fish or frog, but are strongly conserved in mammals.

Animals↗

PubMed2004

An intermediate grade of finished genomic sequence suitable for comparative analyses.

Although the cost of generating draft-quality genomic sequence continues to decline, refining that sequence by the process of "sequence finishing" remains expensive. Near-perfect finished sequence is an appropriate goal for the human genome and a small set of reference genomes; however, such a high-quality product cannot be cost-justified for large numbers of additional genomes, at least for the foreseeable future. Here we describe the generation and quality of an intermediate grade of finished genomic sequence (termed comparative-grade finished sequence), which is tailored for use in multispecies sequence comparisons. Our analyses indicate that this sequence is very high quality (with the residual gaps and errors mostly falling within repetitive elements) and reflects 99% of the total sequence. Importantly, comparative-grade sequence finishing requires approximately 40-fold less reagents and approximately 10-fold less personnel effort compared to the generation of near-perfect finished sequence, such as that produced for the human genome. Although applied here to finishing sequence derived from individual bacterial artificial chromosome (BAC) clones, one could envision establishing routines for refining sequences emanating from whole-genome shotgun sequencing projects to a similar quality level. Our experience to date demonstrates that comparative-grade sequence finishing represents a practical and affordable option for sequence refinement en route to comparative analyses.

Animals↗

PubMed2004

NaCl restriction upregulates renal Slc26a4 through subcellular redistribution: role in Cl- conservation.

Slc26a4 (Pds, pendrin) is an anion transporter expressed in the apical region of type B and non-A, non-B intercalated cells of the distal nephron. It is upregulated by aldosterone analogues and is critical in the development of mineralocorticoid-induced hypertension. Thus, Slc26a4 expression and its role in blood pressure and fluid and electrolyte homeostasis was explored during NaCl restriction, a treatment model in which aldosterone is appropriately increased. Ultrastructural immunolocalization, balance studies, and cortical collecting ducts (CCDs) perfused in vitro were used. With moderate physiological NaCl restriction, Slc26a4 expression in the apical plasma membrane increased 2- to 3-fold in type B intercalated cells. Because Slc26a4 transports Cl-, we tested whether NaCl balance differs in Slc26a4(+/+) and Slc26a4(-/-) mice during NaCl restriction. Cl- absorption was observed in CCDs from Slc26a4(+/+) but not from Slc26a4(-/-) mice. After moderate NaCl restriction, urinary volume and Cl- excretion were increased in Slc26a4(-/-) relative to Slc26a4(+/+) mice. Moreover, Slc26a4(-/-) mice had evidence of relative vascular volume depletion because they had a higher arterial pH, hematocrit, and blood urea nitrogen than wild-type mice. With moderate NaCl restriction, blood pressure was similar in Slc26a4(+/+) and Slc26a4(-/-) mice. However, on a severely restricted intake of NaCl, Slc26a4(-/-) mice were hypotensive relative to wild-type mice. We conclude that Slc26a4 is upregulated with NaCl restriction and is critical in the maintenance of acid-base balance and in the renal conservation of Cl- and water during NaCl restriction.

Acid-Base Equilibrium↗

PubMed2004

Human chromosome 7 circa 2004: a model for structural and functional studies of the human genome.

Human chromosome 7 is arguably amongst the most comprehensively characterized segments of the human genome. By microscopic examination, it belongs to the medium-sized group C submetacentric class, and historical studies involving chromosome-length measurements estimated that it accounts for approximately 5.3% of the human genome (or 160 Mb). Early successes in molecular genetics led to the identification of some of its biomedically important genes, including the T-cell receptor and homeobox families as well as the erythropoietin and cystic fibrosis genes. The Human Genome Project brought chromosome 7-specific and genome-wide initiatives, generating a wealth of genomic resources that have revealed the presence of over 350 disease-associated genes. Two distinct assemblies of the chromosome 7 sequence have been generated-one based largely on mapped large-insert clones and the other based on an integrated whole-genome shotgun sequencing strategy. These two sequences are mainly identical (<1% difference), and both estimate the unit length of chromosome 7 to be just over 158 Mb, remarkably similar to the originally predicted size. Systematic annotation efforts have anchored to the sequence, amongst many features, over 900 known genes and some 1000 other gene structures, as well as over 650 chromosomal breakpoints identified in patients with characterized phenotypic differences. Chromosome 7 has also been shown to contain the highest content of intra-autosomal segmental duplications in the human genome. The orthologous regions of roughly 22 Mb of chromosome 7 are currently being sequenced in multiple other vertebrate species. Examining these comparative sequence data, in conjunction with the other accumulating genomic information about these regions and the rest of the chromosome, should provide a model for the next generation of structural and functional analyses of the human genome.

Chromosomes, Human, Pair 7↗

PubMed2004

Large-scale sequencing of the CD33-related Siglec gene cluster in five mammalian species reveals rapid evolution by multiple mechanisms.

Siglecs are a recently discovered family of animal lectins that belong to the Ig superfamily and recognize sialic acids (Sias). CD33-related Siglecs (CD33rSiglecs) are a subgroup with as-yet-unknown functions, characterized by sequence homology, expression on innate immune cells, conserved cytosolic tyrosine-based signaling motifs, and a clustered localization of their genes. To better understand the biology and evolution of CD33rSiglecs, we sequenced and compared the CD33rSiglec gene cluster from multiple mammalian species. Within the sequenced region, the segments containing CD33rSiglec genes showed a lower degree of sequence conservation. In contrast to the adjacent conserved kallikrein-like genes, the CD33rSiglec genes showed extensive species differences, including expansions of gene subsets; gene deletions, including one human-specific loss of a novel functional primate Siglec (Siglec-13); exon shuffling, generating hybrid genes; accelerated accumulation of nonsynonymous substitutions in the Sia-recognition domain; and multiple instances of mutations of an arginine residue essential for Sia recognition in otherwise intact Siglecs. Nonsynonymous differences between human and chimpanzee orthologs showed uneven distribution between the two beta sheets of the Sia-recognition domain, suggesting biased mutation accumulation. These data indicate that CD33rSiglec genes are undergoing rapid evolution via multiple genetic mechanisms, possibly due to an evolutionary "arms race" between hosts and pathogens involving Sia recognition. These studies, which reflect one of the most complete comparative sequence analyses of a rapidly evolving gene cluster, provide a clearer picture of the ortholog status of CD33rSiglecs among primates and rodents and also facilitate rational recommendations regarding their nomenclature.

Animals↗

PubMed2004

Loss of KCNJ10 protein expression abolishes endocochlear potential and causes deafness in Pendred syndrome mouse model.

BACKGROUND: Pendred syndrome, a common autosomal-recessive disorder characterized by congenital deafness and goiter, is caused by mutations of SLC26A4, which codes for pendrin. We investigated the relationship between pendrin and deafness using mice that have (Slc26a4+/+) or lack a complete Slc26a4 gene (Slc26a4-/-). METHODS: Expression of pendrin and other proteins was determined by confocal immunocytochemistry. Expression of mRNA was determined by quantitative RT-PCR. The endocochlear potential and the endolymphatic K+ concentration were measured with double-barreled microelectrodes. Currents generated by the stria marginal cells were recorded with a vibrating probe. Tissue masses were evaluated by morphometric distance measurements and pigmentation was quantified by densitometry. RESULTS: Pendrin was found in the cochlea in apical membranes of spiral prominence cells and spindle-shaped cells of stria vascularis, in outer sulcus and root cells. Endolymph volume in Slc26a4-/- mice was increased and tissue masses in areas normally occupied by type I and II fibrocytes were reduced. Slc26a4-/- mice lacked the endocochlear potential, which is generated across the basal cell barrier by the K+ channel KCNJ10 localized in intermediate cells. Stria vascularis was hyperpigmented, suggesting unalleviated free radical damage. The basal cell barrier appeared intact; intermediate cells and KCNJ10 mRNA were present but KCNJ10 protein was absent. Endolymphatic K+ concentrations were normal and membrane proteins necessary for K+ secretion were present, including the K+ channel KCNQ1 and KCNE1, Na+/2Cl-/K+ cotransporter SLC12A2 and the gap junction GJB2. CONCLUSIONS: These observations demonstrate that pendrin dysfunction leads to a loss of KCNJ10 protein expression and a loss of the endocochlear potential, which may be the direct cause of deafness in Pendred syndrome.

Animals↗

PubMed2004

Mechanism of iodide/chloride exchange by pendrin.

We performed an electrophysiological study to investigate ion transport of pendrin and thereby understand the pathogenesis of Pendred syndrome. Using pendrin-transfected COS-7 cells, we could show that pendrin transports both iodide and chloride measured as voltage-dependent inward and outward membrane currents. Chloride in the culture medium, [Cl-]o, was efficiently exchanged with cytoplasmic iodide, [I-]i, under physiological concentrations, indicating that pendrin is important for chloride uptake and iodide efflux. Although exchange of iodide in the medium, [I-]o, with cytoplasmic chloride, [Cl-]i, was observed, a significantly high concentration of iodide (10 mm) was required. In addition, either iodide or chloride was required on both sides of the cell membrane for the anion exchange activity of pendrin, indicating that iodide and chloride activate the exchange activity of pendrin while they are transported. The present study further supports that pendrin is responsible for the iodide efflux in thyroid cells where intracellular iodide concentration is high and that the general function of pendrin in other tissues is to transport chloride through exchange with other anions.

Animals↗

PubMed2004

Multi-species sequence comparison reveals dynamic evolution of the elastin gene that has involved purifying selection and lineage-specific insertions/deletions.

BACKGROUND: The elastin gene (ELN) is implicated as a factor in both supravalvular aortic stenosis (SVAS) and Williams Beuren Syndrome (WBS), two diseases involving pronounced complications in mental or physical development. Although the complete spectrum of functional roles of the processed gene product remains to be established, these roles are inferred to be analogous in human and mouse. This view is supported by genomic sequence comparison, in which there are no large-scale differences in the ~1.8 Mb sequence block encompassing the common region deleted in WBS, with the exception of an overall reversed physical orientation between human and mouse. RESULTS: Conserved synteny around ELN does not translate to a high level of conservation in the gene itself. In fact, ELN orthologs in mammals show more sequence divergence than expected for a gene with a critical role in development. The pattern of divergence is non-conventional due to an unusually high ratio of gaps to substitutions. Specifically, multi-sequence alignments of eight mammalian sequences reveal numerous non-aligning regions caused by species-specific insertions and deletions, in spite of the fact that the vast majority of aligning sites appear to be conserved and undergoing purifying selection. CONCLUSIONS: The pattern of lineage-specific, in-frame insertions/deletions in the coding exons of ELN orthologous genes is unusual and has led to unique features of the gene in each lineage. These differences may indicate that the gene has a slightly different functional mechanism in mammalian lineages, or that the corresponding regions are functionally inert. Identified regions that undergo purifying selection reflect a functional importance associated with evolutionary pressure to retain those features.

Animals↗

PubMed2004

Genome sequence of the Brown Norway rat yields insights into mammalian evolution.

The laboratory rat (Rattus norvegicus) is an indispensable tool in experimental medicine and drug development, having made inestimable contributions to human health. We report here the genome sequence of the Brown Norway (BN) rat strain. The sequence represents a high-quality 'draft' covering over 90% of the genome. The BN rat sequence is the third complete mammalian genome to be deciphered, and three-way comparisons with the human and mouse genomes resolve details of mammalian evolution. This first comprehensive analysis includes genes and proteins and their relation to human disease, repeated sequences, comparative genome-wide studies of mammalian orthologous chromosomal regions and rearrangement breakpoints, reconstruction of ancestral karyotypes and the events leading to existing species, rates of variation, and lineage-specific and lineage-independent evolutionary events such as expansion of gene families, orthology relations and protein evolution.

Animals↗

PubMed2004

Identifying gene regulatory elements by genome-wide recovery of DNase hypersensitive sites.

Analysis of the human genome sequence has identified approximately 25000-30000 protein-coding genes, but little is known about how most of these are regulated. Mapping DNase I hypersensitive (HS) sites has traditionally represented the gold-standard experimental method for identifying regulatory elements, but the labor-intensive nature of this technique has limited its application to only a small number of human genes. We have developed a protocol to generate a genome-wide library of gene regulatory sequences by cloning DNase HS sites. We generated a library of DNase HS sites from quiescent primary human CD4(+) T cells and analyzed approximately 5600 of the resulting clones. Compared to sequences from randomly generated in silico libraries, sequences from these clones were found to map more frequently to regions of the genome known to contain regulatory elements, such as regions upstream of genes, within CpG islands, and in sequences that align between mouse and human. These cloned sites also tend to map near genes that have detectable transcripts in CD4(+) T cells, demonstrating that transcriptionally active regions of the genome are being selected. Validation of putative regulatory elements was achieved by repeated recovery of the same sequence and real-time PCR. This cloning strategy, which can be scaled up and applied to any cell line or tissue, will be useful in identifying regulatory elements controlling global expression differences that delineate tissue types, stages of development, and disease susceptibility.

CD4-Positive T-Lymphocytes↗

PubMed2004

Results of a genome-wide linkage scan for stuttering.

We performed a linkage study of stuttering using 392 markers distributed across the genome in a series of 68 families identified in the general outbred population of North America and Europe. Standardized diagnosis was performed using recorded samples of both conversation and reading, in which stuttering dysfluencies were scored as percentage of dysfluent words and syllables. Analysis was first performed using non-parametric methods implemented in GENEHUNTER, where we obtained maximum statistical support for markers of chromosome 18, with a maximum NPL (Sall) of 1.51 at D18S976. The single largest pedigree within our sample (pedigree 0006) alone gave an NPL of 4.72 at D18S976. For fine mapping, we analyzed 18 markers on chromosome 18 across all families using ALLEGRO. Overall NPL (Srobdom) scores >5 were obtained with markers on 18p, and Z(lr) scores >/=2.5 on 18p and proximal 18q. Furthermore, pedigree 0006 alone gave an NPL (Srobdom) of 5.35. Overall our results suggest chromosome 18 may harbor a predisposing locus for this disorder, and additional genes may exist.

Chromosome Mapping↗

PubMed2004

Comparative sequence analysis of the Gdf6 locus reveals a duplicon-mediated chromosomal rearrangement in rodents and rapidly diverging coding and regulatory sequences.

Duplicated segments of genomic DNA can catalyze both gene evolution and chromosome evolution. Here we describe a rodent-specific duplication involving the Uqcrb gene, a cis-regulatory element for the Gdf6 gene, and a chromosomal rearrangement. Comparisons of Gdf6 sequences from several placental mammals and platypus revealed many strongly conserved regions flanking Gdf6 and the adjacent Uqcrb gene. However, in rat and mouse a synteny break resides approximately 70 kb upstream of Gdf6, such that Gdf6 and Uqcrb are on separate chromosomes. In rodents, Gdf6 and Uqcrb are both associated with homologous duplicons that may have catalyzed a rearrangement separating the two genes. However, the duplicon spanned both Uqcrb and a cis-regulatory element that controls Gdf6 transcription in limb skeletal joints. In mouse and rat, one duplicon now contains a degrading Uqcrb pseudogene but retains strongly conserved sequences within a Gdf6 enhancer. In contrast, the other duplicon has retained the intact Uqcrb gene and (in mouse) a copy of the Gdf6 enhancer that has acquired novel mutations. The duplicons have separately maintained distinct functions of the ancestral sequence, consistent with a "subfunction partitioning" evolutionary model. These findings also provide an example of a duplication that mobilized a tissue-specific enhancer from its cognate gene, and new evidence that duplications can be associated with chromosomal rearrangements. Furthermore, these data suggest that segmental duplications could lead to evolution of novel gene expression patterns via diversification of regulatory elements.

Animals↗

PubMed2004

Aligning multiple genomic sequences with the threaded blockset aligner.

We define a "threaded blockset," which is a novel generalization of the classic notion of a multiple alignment. A new computer program called TBA (for "threaded blockset aligner") builds a threaded blockset under the assumption that all matching segments occur in the same order and orientation in the given sequences; inversions and duplications are not addressed. TBA is designed to be appropriate for aligning many, but by no means all, megabase-sized regions of multiple mammalian genomes. The output of TBA can be projected onto any genome chosen as a reference, thus guaranteeing that different projections present consistent predictions of which genomic positions are orthologous. This capability is illustrated using a new visualization tool to view TBA-generated alignments of vertebrate Hox clusters from both the mammalian and fish perspectives. Experimental evaluation of alignment quality, using a program that simulates evolutionary change in genomic sequences, indicates that TBA is more accurate than earlier programs. To perform the dynamic-programming alignment step, TBA runs a stand-alone program called MULTIZ, which can be used to align highly rearranged or incompletely sequenced genomes. We describe our use of MULTIZ to produce the whole-genome multiple alignments at the Santa Cruz Genome Browser.

Animals↗

PubMed2004

The status, quality, and expansion of the NIH full-length cDNA project: the Mammalian Gene Collection (MGC).

The National Institutes of Health's Mammalian Gene Collection (MGC) project was designed to generate and sequence a publicly accessible cDNA resource containing a complete open reading frame (ORF) for every human and mouse gene. The project initially used a random strategy to select clones from a large number of cDNA libraries from diverse tissues. Candidate clones were chosen based on 5'-EST sequences, and then fully sequenced to high accuracy and analyzed by algorithms developed for this project. Currently, more than 11,000 human and 10,000 mouse genes are represented in MGC by at least one clone with a full ORF. The random selection approach is now reaching a saturation point, and a transition to protocols targeted at the missing transcripts is now required to complete the mouse and human collections. Comparison of the sequence of the MGC clones to reference genome sequences reveals that most cDNA clones are of very high sequence quality, although it is likely that some cDNAs may carry missense variants as a consequence of experimental artifact, such as PCR, cloning, or reverse transcriptase errors. Recently, a rat cDNA component was added to the project, and ongoing frog (Xenopus) and zebrafish (Danio) cDNA projects were expanded to take advantage of the high-throughput MGC pipeline.

Animals↗